Thermophilic and thermoacidophilic sugar transporter genes and enzymes from Alicyclobacillus acidocaldarius and related organisms, methods

ABSTRACT

Isolated and/or purified polypeptides and nucleic acid sequences encoding polypeptides from  Alicyclobacillus acidocaldarius  are provided. Further provided are methods for transporting sugars across cell membranes using isolated and/or purified polypeptides and nucleic acid sequences from  Alicyclobacillus acidocaldarius.

RELATED APPLICATIONS

This application is a divisional of U.S. patent application Ser. No.13/200,164, filed Sep. 20, 2011, pending, which is a divisional of U.S.patent application Ser. No. 13/066,645, filed Apr. 19, 2011, now U.S.Pat. No. 8,071,748, issued Dec. 6, 2011, which is a divisional of U.S.patent application Ser. No. 12/380,554, filed Feb. 26, 2009, now U.S.Pat. No. 7,960,534, issued Jun. 14, 2011, which application claims thebenefit of the filing date of U.S. Provisional Patent Application Ser.No. 61/031,593, filed Feb. 26, 2008, for “THERMOPHILIC ANDTHERMOACIDOPHILIC SUGAR TRANSPORTER GENES AND ENZYMES FROMALICYCLOBACILLUS ACIDOCALDARIUS AND RELATED ORGANISMS, METHODS,” thedisclosure of each of which is hereby incorporated herein by thisreference in its entirety.

STATEMENT ACCORDING TO 37 C.F.R. §1.821(c) OR (e)—SEQUENCE LISTINGSUBMITTED AS ASCII TEXT FILE

Pursuant to 37 C.F.R. §1.821(c) or (e), a file containing an PDF versionof the Sequence Listing has been submitted concomitant with thisapplication, the contents of which are hereby incorporated by reference.

GOVERNMENT RIGHTS

This invention was made with government support under Contract No.DE-AC07-99ID13727 and Contract No. DE-AC07-05ID14517 awarded by theUnited States Department of Energy. The government has certain rights inthe invention.

TECHNICAL FIELD

The present invention relates generally to biotechnology. Morespecifically, the present invention relates to isolated and/or purifiedpolypeptides and nucleic acid sequences encoding polypeptides fromAlicyclobacillus acidocaldarius and methods for their use.

BACKGROUND

Dilute acid hydrolysis to remove hemicellulose from lignocellulosicmaterials is one of the most developed pretreatment techniques forlignocellulose and is currently favored (Hamelinck et al., 2005) becauseit results in fairly high yields of xylose (75% to 90%). Conditions thatare typically used range from 0.1% to 1.5% sulfuric acid andtemperatures above 160° C. The high temperatures used result insignificant levels of thermal decomposition products that inhibitsubsequent microbial fermentations (Lavarack et al., 2002). Hightemperature hydrolysis requires pressurized systems, steam generation,and corrosion resistant materials in reactor construction due to themore corrosive nature of acid at elevated temperatures.

Low temperature acid hydrolyses are of interest because they have thepotential to overcome several of the above shortcomings (Tsao et al.,1987). It has been demonstrated that 90% of hemicellulose can besolubilized as oligomers in a few hours of acid treatment in thetemperature range of 80° C. to 100° C. It has also been demonstratedthat the sugars produced in low temperature acid hydrolysis are stableunder those same conditions for at least 24 hours with no detectabledegradation to furfural decomposition products. Finally, sulfuric acidtypically used in pretreatments is not as corrosive at lowertemperatures. The use of lower temperature acid pretreatments requiresmuch longer reaction times to achieve acceptable levels of hydrolysis.Although 90% hemicellulose solubilization has been shown (Tsao, 1987),the bulk of the sugars are in the form of oligomers and are not in themonomeric fowl. The organisms currently favored in subsequentfermentation steps cannot utilize sugar oligomers (Garrote et al., 2001)and the oligomer-containing hydrolysates require further processing tomonomers, usually as a second acid or alkaline hydrolysis step (Garroteet al., 2001).

Other acidic pretreatment methods include autohydrolysis and hot waterwashing. In autohydrolysis, biomass is treated with steam at hightemperatures (˜240° C.), which cleaves acetyl side chains associatedwith hemicellulose to produce acetic acid that functions in a similarmanner to sulfuric acid in acid hydrolysis. Higher pretreatmenttemperatures are required as compared to dilute acid hydrolysis becauseacetic acid is a much weaker acid than sulfuric. At temperatures below240° C., the hemicellulose is not completely hydrolyzed to sugarmonomers and has high levels of oligomers (Garrote et al., 2001). In hotwater washing, biomass is contacted with water (under pressure) atelevated temperatures 160° C. to 220° C. This process can effectivelyhydrolyze greater than 90% of the hemicellulose present and thesolubilized hemicellulose was typically over 95% in the form ofoligomers (Liu and Wyman, 2003).

BRIEF SUMMARY OF THE INVENTION

Embodiments of the invention relate to purified and/or isolatednucleotide sequences of the genome of Alicyclobacillus acidocaldarius,or a homologue or fragment thereof. In one embodiment of the invention,the nucleotide sequence is selected from at least one of SEQ ID NOS:2,19, 36, 53, 70, 87, 104, 121, 138, 155, 172, 189, 206, 223, 240, 257,274, 291, 308, 325, 342, 359, 376, 393, 410, 427, 444, 461, 478, 495,512, 529, 546, 563, 580, 597, 614, 631, 648, 665, 682, 699, 716, 733,750, and 767 or a homologue or fragment thereof. In another embodimentof the invention, the homologue is selected from the group consisting ofa nucleotide sequence having at least 80% sequence identity to at leastone of SEQ ID NOS:2, 19, 36, 53, 70, 87, 104, 121, 138, 155, 172, 189,206, 223, 240, 257, 274, 291, 308, 325, 342, 359, 376, 393, 410, 427,444, 461, 478, 495, 512, 529, 546, 563, 580, 597, 614, 631, 648, 665,682, 699, 716, 733, 750, and 767.

Embodiments of the invention may further relate to an isolated and/orpurified nucleic acid sequence comprising a nucleic acid sequenceencoding a polypeptide selected from the group consisting of apolypeptide having at least 90% sequence identity to at least one of SEQID NOS:1, 18, 35, 52, 69, 86, 103, 120, 137, 154, 171, 188, 205, 222,239, 256, 273, 290, 307, 324, 341, 358, 375, 392, 409, 426, 443, 460,477, 494, 511, 528, 545, 562, 579, 596, 613, 630, 647, 664, 681, 698,715, 732, 749, and 766.

Embodiments of the invention also relate to isolated and/or purifiedpolypeptides coded for by a nucleotide sequence comprising a nucleotidesequence of the genome of Alicyclobacillus acidocaldarius, or ahomologue or fragment thereof. In one embodiment, the nucleotidesequence comprises a nucleotide sequence selected from the groupconsisting of a nucleotide sequence having at least 80% sequenceidentity to at least one of SEQ ID NOS:2, 19, 36, 53, 70, 87, 104, 121,138, 155, 172, 189, 206, 223, 240, 257, 274, 291, 308, 325, 342, 359,376, 393, 410, 427, 444, 461, 478, 495, 512, 529, 546, 563, 580, 597,614, 631, 648, 665, 682, 699, 716, 733, 750, and 767.

In another embodiment of the invention, the nucleotide sequencecomprises a nucleotide sequence selected from at least one of SEQ IDNOS:2, 19, 36, 53, 70, 87, 104, 121, 138, 155, 172, 189, 206, 223, 240,257, 274, 291, 308, 325, 342, 359, 376, 393, 410, 427, 444, 461, 478,495, 512, 529, 546, 563, 580, 597, 614, 631, 648, 665, 682, 699, 716,733, 750, and 767 or a homologue or fragment thereof. In still anotherembodiment, the polypeptide comprises an amino acid sequence of SEQ IDNOS:1, 18, 35, 52, 69, 86, 103, 120, 137, 154, 171, 188, 205, 222, 239,256, 273, 290, 307, 324, 341, 358, 375, 392, 409, 426, 443, 460, 477,494, 511, 528, 545, 562, 579, 596, 613, 630, 647, 664, 681, 698, 715,732, 749, and 766. In yet another embodiment, the polypeptide comprisesan amino acid sequence selected from the group consisting of apolypeptide having at least 90% sequence identity to at least one of SEQID NOS:1, 18, 35, 52, 69, 86, 103, 120, 137, 154, 171, 188, 205, 222,239, 256, 273, 290, 307, 324, 341, 358, 375, 392, 409, 426, 443, 460,477, 494, 511, 528, 545, 562, 579, 596, 613, 630, 647, 664, 681, 698,715, 732, 749, and 766.

In embodiments of the invention, the polypeptides may be acidophilicand/or thermophilic. In further embodiments, the polypeptides may beglycosylated, pegylated, and/or otherwise post-translationally modified.

Embodiments of methods include providing a recombinant, purified, and/orisolated polypeptide selected from the group consisting of a polypeptidehaving at least 90% sequence identity to SEQ ID NOS:1, 18, 35, 52, 69,86, 103, 120, 137, 154, 171, 188, 205, 222, 239, 256, 273, 290, 307,324, 341, 358, 375, 392, 409, 426, 443, 460, 477, 494, 511, 528, 545,562, 579, 596, 613, 630, 647, 664, 681, 698, 715, 732, 749, and 766 inor associated with a cell membrane and transporting a sugar across thecell membrane using the a recombinant, purified, and/or isolatedpolypeptide in conjunction with other cellular components.

Further embodiments of methods include placing a cell producing orencoding a recombinant, purified, and/or isolated nucleotide sequencecomprising a nucleotide sequence selected from the group consisting of anucleotide sequences having at least 90% sequence identity to at leastone of the sequences of SEQ ID NOS:2, 19, 36, 53, 70, 87, 104, 121, 138,155, 172, 189, 206, 223, 240, 257, 274, 291, 308, 325, 342, 359, 376,393, 410, 427, 444, 461, 478, 495, 512, 529, 546, 563, 580, 597, 614,631, 648, 665, 682, 699, 716, 733, 750, and 767 and/or a recombinant,purified, and/or isolated polypeptide selected from the group consistingof a polypeptide having at least 90% sequence identity to at least oneof the sequences of SEQ ID NOS:1, 18, 35, 52, 69, 86, 103, 120, 137,154, 171, 188, 205, 222, 239, 256, 273, 290, 307, 324, 341, 358, 375,392, 409, 426, 443, 460, 477, 494, 511, 528, 545, 562, 579, 596, 613,630, 647, 664, 681, 698, 715, 732, 749, and 766 in a environmentcomprising temperatures at or above about 25, 30, 35, 40, 45, 50, 55,60, 65, 70, 75, 80, 85, 90, and/or 95 degrees Celsius and/or a pH at,below, and/or above 8, 7, 6, 5, 4, 3, 2, 1, and/or 0.

These and other aspects of the invention will become apparent to theskilled artisan in view of the teachings contained herein.

BRIEF DESCRIPTION OF THE SEVERAL VIEWS OF THE DRAWINGS

FIG. 1 depicts a sequence alignment between SEQ ID NO:1 (RAAC00572) andemb|CAB65652.1|, ref|NP_(—)623417.1|, ref|YP_(—)001662812.1|,ref|YP_(—)001179257.1|, and ref|YP_(—)699602.1|(SEQ ID NOS:3-7),respectively, which all have the function assigned to SEQ ID NO:1 inTable 1. Amino acids conserved among all sequences are indicated by a“*” and generally conserved amino acids are indicated by a “:”.

FIGS. 2A and 2B depict a sequence alignment between SEQ ID NO:18(RAAC00573) and emb|CAB65651.1|, pdb|1URG|A, pdb|1URD|A,ref|YP_(—)001662811.1|, and ref|NP_(—)623418.1|(SEQ ID NOS:20-24),respectively, which all have the function assigned to SEQ ID NO:18 inTable 1. Amino acids conserved among all sequences are indicated by a“*” and generally conserved amino acids are indicated by a “:”.

FIGS. 3A and 3B depict a sequence alignment between SEQ ID NO:35(RAAC00608) and ref|NP_(—)391276.1|, ref|YP_(—)001422694.1|,ref|NP_(—)347967.1|, ref|ZP_(—)01886765.1|, and ref|YP_(—)804553.1| (SEQID NOS:37-41), respectively, which all have the function assigned to SEQID NO:35 in Table 1. Amino acids conserved among all sequences areindicated by a “*” and generally conserved amino acids are indicated bya “:”.

FIGS. 4A and 4B depict a sequence alignment between SEQ ID NO:52(RAAC00626) and ref|YP_(—)001108359.1|, ref|YP_(—)001662045.1|,ref|YP_(—)147976.1|, ref|YP_(—)001126119.1|, and ref|YP_(—)001409972.1|(SEQ ID NOS:54-58), respectively, which all have the function assignedto SEQ ID NO:52 in Table 1. Amino acids conserved among all sequencesare indicated by a “*” and generally conserved amino acids are indicatedby a “:”.

FIG. 5 depicts a sequence alignment between SEQ ID NO:69 (RAAC00627) andref|YP_(—)001108360.1|, ref|ZP_(—)01730302.1|, ref|YP_(—)001662044.1|,ref|YP_(—)171303.1|, and ref|YP_(—)922080.1| (SEQ ID NOS:71-75),respectively, which all have the function assigned to SEQ ID NO:69 inTable 1. Amino acids conserved among all sequences are indicated by a“*” and generally conserved amino acids are indicated by a “:”.

FIG. 6 depicts a sequence alignment between SEQ ID NO:86 (RAAC00628) andref|YP_(—)001108361.1|, ref|YP_(—)001662043.1|, ref|YP_(—)147974.1|,ref|YP_(—)001126117.1|, and ref|NP_(—)694394.1| (SEQ ID NOS:88-92),respectively, which all have the function assigned to SEQ ID NO:86 inTable 1. Amino acids conserved among all sequences are indicated by a“*” and generally conserved amino acids are indicated by a “:”.

FIGS. 7A and 7B depict a sequence alignment between SEQ ID NO:103(RAAC00662) and ref|YP_(—)644805.1|, ref|YP_(—)589403.1|,ref|YP_(—)822512.1|, ref|YP_(—)825097.1|, and ref|YP_(—)001108350.1|(SEQID NOS:105-109), respectively, which all have the function assigned toSEQ ID NO:103 in Table 1. Amino acids conserved among all sequences areindicated by a “*” and generally conserved amino acids are indicated bya “:”.

FIG. 8 depicts a sequence alignment between SEQ ID NO:120 (RAAC00732)and emb|CAE45698.1|, ref|ZP_(—)02329051.1|, ref|YP_(—)001181115.1|,ref|NP_(—)623554.1|, and ref|YP_(—)001664561.1| (SEQ ID NOS:122-126),respectively, which all have the function assigned to SEQ ID NO:120 inTable 1. Amino acids conserved among all sequences are indicated by a“*” and generally conserved amino acids are indicated by a “:”.

FIGS. 9A and 9B depict a sequence alignment between SEQ ID NO:137(RAAC00804) and ref|NP_(—)623507.1|, ref|YP_(—)001469682.1|,ref|YP_(—)145275.1|, ref|YP_(—)006204.1|, and ref|YP_(—)001432292.1|(SEQ ID NOS:139-143), respectively, which all have the function assignedto SEQ ID NO:137 in Table 1. Amino acids conserved among all sequencesare indicated by a “*” and generally conserved amino acids are indicatedby a “:”.

FIGS. 10A and 10B depict a sequence alignment between SEQ ID NO:154(RAAC00824) and ref|ZP_(—)02128903.1|, ref|YP_(—)001243927.1|,ref|YP_(—)001390862.1|, ref|YP_(—)001254028.1|, and ref|NP_(—)228405.1|(SEQ ID NOS:156-160), respectively, which all have the function assignedto SEQ ID NO:154 in Table 1. Amino acids conserved among all sequencesare indicated by a “*” and generally conserved amino acids are indicatedby a “:”.

FIGS. 11A and 11B depict a sequence alignment between SEQ ID NO:171(RAAC01073) and ref|YP_(—)804553.1|, ref|NP_(—)391276.1|,ref|YP_(—)001422694.1|, ref|NP 347967.1|, and ref|NP_(—)978526.1|(SEQ IDNOS:173-177), respectively, which all have the function assigned to SEQID NO:171 in Table 1. Amino acids conserved among all sequences areindicated by a “*” and generally conserved amino acids are indicated bya “:”.

FIGS. 12A and 12B depict a sequence alignment between SEQ ID NO:188(RAAC01120) and ref|YP_(—)001089934.1|, ref|ZP_(—)01801573.1|,ref|ZP_(—)01173000.1|, ref|YP_(—)076008.1|, and gb|ABP_(—)57783.1| (SEQID NOS:190-194), respectively, which all have the function assigned toSEQ ID NO:188 in Table 1. Amino acids conserved among all sequences areindicated by a “*” and generally conserved amino acids are indicated bya “:”.

FIG. 13 depicts a sequence alignment between SEQ ID NO:205 (RAAC01122)and ref|YP_(—)001527659.1|, ref|YP_(—)321082.1|, ref|NP_(—)484832.1|,ref|ZP_(—)01631485.1|, and gb|AAD33665.1|AF135398_(—)2 (SEQ IDNOS:207-211), respectively, which all have the function assigned to SEQID NO:205 in Table 1. Amino acids conserved among all sequences areindicated by a “*” and generally conserved amino acids are indicated bya “:”.

FIG. 14 depicts a sequence alignment between SEQ ID NO:222 (RAAC01168)and ref|ZP_(—)02083881.1|, ref|ZP_(—)02075264.1|, ref|ZP_(—)01461962.1|,ref|YP_(—)632791.1|, and ref|NP_(—)348945.1| (SEQ ID NOS:224-228),respectively, which all have the function assigned to SEQ ID NO:222 inTable 1. Amino acids conserved among all sequences are indicated by a“*” and generally conserved amino acids are indicated by a “:”.

FIG. 15 depicts a sequence alignment between SEQ ID NO:239 (RAAC01169)and ref|ZP_(—)02075265.1|, ref|ZP_(—)02083878.1|, ref|ZP_(—)02085002.1|,ref|ZP_(—)01978997.1|, and ref|ZP_(—)02045164.1| (SEQ ID NOS:241-245),respectively, which all have the function assigned to SEQ ID NO:239 inTable 1. Amino acids conserved among all sequences are indicated by a“*” and generally conserved amino acids are indicated by a “:”.

FIG. 16 depicts a sequence alignment between SEQ ID NO:256 (RAAC01276)and ref|YP_(—)173899.1|, ref|NP 241985.1|, ref|ZP_(—)01168682.1|,ref|ZP_(—)02087132.1|, and ref|YP_(—)173890.1| (SEQ ID NOS:258-262),respectively, which all have the function assigned to SEQ ID NO:256 inTable 1. Amino acids conserved among all sequences are indicated by a“*” and generally conserved amino acids are indicated by a “:”.

FIG. 17 depicts a sequence alignment between SEQ ID NO:273 (RAAC01277)and ref|YP_(—)949009.1|, ref|YP_(—)73889.1|, ref|YP_(—)001361891.1|,ref|YP_(—)832803.1|, and ref|ZP_(—)01168681.1| (SEQ ID NOS:275-279),respectively, which all have the function assigned to SEQ ID NO:273 inTable 1. Amino acids conserved among all sequences are indicated by a“*” and generally conserved amino acids are indicated by a “:”.

FIGS. 18A and 18B depict a sequence alignment between SEQ ID NO:290(RAAC01278) and ref|YP_(—)289760.1|, ref|NP_(—)241983.1|,ref|YP_(—)173897.1|, ref|YP_(—)173888.1|, and ref|NP_(—)357484.21 (SEQID NOS:292-296), respectively, which all have the function assigned toSEQ ID NO:290 in Table 1. Amino acids conserved among all sequences areindicated by a “*” and generally conserved amino acids are indicated bya “:”.

FIG. 19 depicts a sequence alignment between SEQ ID NO:307 (RAAC01279)and ref|NP_(—)693016.1|, ref|ZP_(—)01924075.1|, ref|YP_(—)74646.1|,ref|ZP_(—)01854967.1|, and ref|ZP_(—)01088898.1| (SEQ ID NOS:309-313),respectively, which all have the function assigned to SEQ ID NO:307 inTable 1. Amino acids conserved among all sequences are indicated by a“*” and generally conserved amino acids are indicated by a “:”.

FIGS. 20A and 20B depict a sequence alignment between SEQ ID NO:324(RAAC01316) and ref|YP_(—)001523023.1|, ref|ZP_(—)01746012.1|,ref|YP_(—)001479789.1|, ref|YP_(—)001585330.1|, and ref|NP_(—)845565.1|(SEQ ID NOS:326-330), respectively, which all have the function assignedto SEQ ID NO:324 in Table 1. Amino acids conserved among all sequencesare indicated by a “*” and generally conserved amino acids are indicatedby a “:”.

FIG. 21 depicts a sequence alignment between SEQ ID NO:341 (RAAC01502)and ref|YP_(—)001113858.1|, ref|ZP_(—)02171111.1|,ref|YP_(—)001488480.1|, ref|YP_(—)878667.1|, and ref|NP_(—)693494.1|(SEQ ID NOS:343-347), respectively, which all have the function assignedto SEQ ID NO:341 in Table 1. Amino acids conserved among all sequencesare indicated by a “*” and generally conserved amino acids are indicatedby a “:”.

FIG. 22 depicts a sequence alignment between SEQ ID NO:358 (RAAC01599)and ref|YP_(—)114375.1|, ref|YP_(—)001568000.1|, ref|YP_(—)001254026.1|,ref|YP_(—)001390860.1|, and ref|YP_(—)001275707.1|(SEQ ID NOS:360-364),respectively, which all have the function assigned to SEQ ID NO:358 inTable 1. Amino acids conserved among all sequences are indicated by a“*” and generally conserved amino acids are indicated by a “:”.

FIGS. 23A and 23B depict a sequence alignment between SEQ ID NO:375(RAAC01600) and ref YP_(—)076008.1|, ref|YP_(—)001624170.1|,ref|YP_(—)001089934.1|, ref|ZP_(—)01801573.1|, and ref|ZP_(—)01850509.1|(SEQ ID NOS:377-381), respectively, which all have the function assignedto SEQ ID NO:375 in Table 1. Amino acids conserved among all sequencesare indicated by a “*” and generally conserved amino acids are indicatedby a “:”.

FIG. 24 depicts a sequence alignment between SEQ ID NO:392 (RAAC01625)and ref|YP_(—)001527658.1|, ref|ZP_(—)01189621.1|,ref|YP_(—)001327980.1|, ref|NP_(—)436764.1|, and ref|NP 463711.1| (SEQID NOS:394-398), respectively, which all have the function assigned toSEQ ID NO:392 in Table 1. Amino acids conserved among all sequences areindicated by a “*” and generally conserved amino acids are indicated bya “:”.

FIGS. 25A and 25B depict a sequence alignment between SEQ ID NO:409(RAAC01626) and ref|YP_(—)001624853.1|, ref|YP_(—)832862.1|,ref|NP_(—)733496.1|, ref|NP_(—)961026.1|, and ref|YP_(—)881303.1|(SEQ IDNOS:411-415), respectively, which all have the function assigned to SEQID NO:409 in Table 1. Amino acids conserved among all sequences areindicated by a “*” and generally conserved amino acids are indicated bya “:”.

FIG. 26 depicts a sequence alignment between SEQ ID NO:426 (RAAC01627)and ref|ZP_(—)01115262.1|, ref|NP_(—)815894.1|, ref|YP_(—)173820.1|,ref|NP_(—)624601.1|, and ref|NP 336858.1| (SEQ ID NOS:428-432),respectively, which all have the function assigned to SEQ ID NO:426 inTable 1. Amino acids conserved among all sequences are indicated by a“*” and generally conserved amino acids are indicated by a “:”.

FIGS. 27A and 27B depict a sequence alignment between SEQ ID NO:443(RAAC01754) and ref|ZP_(—)01172341.1|, ref|YP_(—)001665505.1|,ref|YP_(—)001663811.1|, ref|YP_(—)001124945.1|, and ref|NP_(—)622450.1|(SEQ ID NOS:445-449), respectively, which all have the function assignedto SEQ ID NO:443 in Table 1. Amino acids conserved among all sequencesare indicated by a “*” and generally conserved amino acids are indicatedby a “:”.

FIGS. 28A and 28B depict a sequence alignment between SEQ ID NO:460(RAAC01756) and ref|YP_(—)001665503.1|, ref|NP_(—)622452.1|,ref|YP_(—)001179269.1|, ref|YP_(—)001124947.1|, and ref|NP 244557.1|(SEQ ID NOS:462-466), respectively, which all have the function assignedto SEQ ID NO:460 in Table 1. Amino acids conserved among all sequencesare indicated by a “*” and generally conserved amino acids are indicatedby a “:”.

FIG. 29 depicts a sequence alignment between SEQ ID NO:477 (RAAC01757)and ref|YP_(—)001665502.1|, ref|YP_(—)001663808.1|, ref|NP_(—)622453.1|,ref|ZP_(—)01172344.1|, and ref|YP_(—)001179270.1| (SEQ ID NOS:479-483),respectively, which all have the function assigned to SEQ ID NO:477 inTable 1. Amino acids conserved among all sequences are indicated by a“*” and generally conserved amino acids are indicated by a “:”.

FIG. 30 depicts a sequence alignment between SEQ ID NO:494 (RAAC01758)and ref|NP_(—)622454.1|, ref|YP_(—)001665501.1|, ref|YP_(—)001663807.1|,ref|YP_(—)001179271.1|, and ref|YP_(—)001124949.1| (SEQ ID NOS:496-500),respectively, which all have the function assigned to SEQ ID NO:494 inTable 1. Amino acids conserved among all sequences are indicated by a“*” and generally conserved amino acids are indicated by a “:”.

FIGS. 31A and 31B depict a sequence alignment between SEQ ID NO:511(RAAC01989) and ref|YP_(—)001614945.1|, ref|YP_(—)001619074.1|,ref|YP_(—)001618197.1|, ref|YP_(—)001471644.1|, andref|YP_(—)001613999.1| (SEQ ID NOS:513-517), respectively, which allhave the function assigned to SEQ ID NO:511 in Table 1. Amino acidsconserved among all sequences are indicated by a “*” and generallyconserved amino acids are indicated by a “:”.

FIG. 32 depicts a sequence alignment between SEQ ID NO:528 (RAAC01990)and ref|YP_(—)829900.1|, ref|YP_(—)947699.1|, ref|YP_(—)001032750.1|,ref|YP_(—)001614944.1|, and ref|YP_(—)001545164.1| (SEQ ID NOS:530-534),respectively, which all have the function assigned to SEQ ID NO:528 inTable 1. Amino acids conserved among all sequences are indicated by a“*” and generally conserved amino acids are indicated by a “:”.

FIG. 33 depicts a sequence alignment between SEQ ID NO:545 (RAAC01991)and ref|YP_(—)001032749.1|, ref|YP_(—)001567539.1|, ref|YP_(—)614737.1|,ref|YP_(—)001471642.1|, and ref|YP_(—)063070.1| (SEQ ID NOS:547-551),respectively, which all have the function assigned to SEQ ID NO:545 inTable 1. Amino acids conserved among all sequences are indicated by a“*” and generally conserved amino acids are indicated by a “:”.

FIG. 34 depicts a sequence alignment between SEQ ID NO:562 (RAAC01992)and ref|YP_(—)001614942.1|, ref|YP_(—)001545162.1|,ref|ZP_(—)01473687.1|, ref|NP_(—)798861.1|, and ref|ZP_(—)01262242.1|(SEQ ID NOS:564-568), respectively, which all have the function assignedto SEQ ID NO:562 in Table 1. Amino acids conserved among all sequencesare indicated by a “*” and generally conserved amino acids are indicatedby a “:”.

FIG. 35 depicts a sequence alignment between SEQ ID NO:579 (RAAC02175)and ref|YP_(—)644454.1|, ref|ZP_(—)00133639.21, ref|YP_(—)001337166.1|,ref|YP_(—)001591175.1|, and ref|YP_(—)355005.1| (SEQ ID NOS:581-585),respectively, which all have the function assigned to SEQ ID NO:579 inTable 1. Amino acids conserved among all sequences are indicated by a“*” and generally conserved amino acids are indicated by a “:”.

FIG. 36 depicts a sequence alignment between SEQ ID NO:596 (RAAC02176)and ref|ZP_(—)01440479.1|, gb|EDR95515.1|, ref|YP_(—)037038.1|,ref|ZP_(—)02260050.1|, and ref|YP_(—)029022.1| (SEQ ID NOS:598-602),respectively, which all have the function assigned to SEQ ID NO:596 inTable 1. Amino acids conserved among all sequences are indicated by a“*” and generally conserved amino acids are indicated by a “:”.

FIG. 37 depicts a sequence alignment between SEQ ID NO:613 (RAACO02177)and ref|NP_(—)832709.1|, ref|YP_(—)001565594.1|, ref|ZP_(—)00239479.1|,ref|YP_(—)001178244.1|, and ref|YP_(—)001337168.1|(SEQ ID NOS:615-619),respectively, which all have the function assigned to SEQ ID NO:613 inTable 1. Amino acids conserved among all sequences are indicated by a“*” and generally conserved amino acids are indicated by a “:”.

FIGS. 38A and 38B depict a sequence alignment between SEQ ID NO:630(RAAC02613) and ref|YP_(—)001546405.1|, ref|ZP_(—)02171436.1|,ref|ZP_(—)02295559.1|, ref|YP_(—)765572.1|, and ref|NP 106947.1| (SEQ IDNOS:632-636), respectively, which all have the function assigned to SEQID NO:630 in Table 1. Amino acids conserved among all sequences areindicated by a “*” and generally conserved amino acids are indicated bya “:”.

FIG. 39 depicts a sequence alignment between SEQ ID NO:647 (RAAC02614)and gb|AAD33665.1|AF135398_(—)2, dbj|BAA28360.1|, ref|ZP_(—)02016963.1|,ref|YP_(—)765571.1|, and ref|NP_(—)357451.21 (SEQ ID NOS:649-653),respectively, which all have the function assigned to SEQ ID NO:647 inTable 1. Amino acids conserved among all sequences are indicated by a“*” and generally conserved amino acids are indicated by a “:”.

FIG. 40 depicts a sequence alignment between SEQ ID NO:664 (RAAC02615)and ref|ZP_(—)02171438.1|, ref|ZP_(—)02016964.1|, ref|ZP_(—)01188237.1|,ref|YP_(—)176793.1|, and ref|YP_(—)001546403.1|(SEQ ID NOS:666-670),respectively, which all have the function assigned to SEQ ID NO:664 inTable 1. Amino acids conserved among all sequences are indicated by a“*” and generally conserved amino acids are indicated by a “:”.

FIG. 41 depicts a sequence alignment between SEQ ID NO:681 (RAAC02733)and ref|YP_(—)001179080.1|, ref|NP_(—)244315.1|, ref|YP_(—)147766.1|,ref|YP_(—)001125909.1|, and ref|YP_(—)077677.1| (SEQ ID NOS:683-687),respectively, which all have the function assigned to SEQ ID NO:681 inTable 1. Amino acids conserved among all sequences are indicated by a“*” and generally conserved amino acids are indicated by a “:”.

FIG. 42 depicts a sequence alignment between SEQ ID NO:698 (RAAC02734)and ref|YP_(—)001179079.1|, ref|YP_(—)001036823.1|,ref|YP_(—)001312352.1|, ref|NP 437024.1|, and ref|YP_(—)471845.1| (SEQID NOS:700-704), respectively, which all have the function assigned toSEQ ID NO:968 in Table 1. Amino acids conserved among all sequences areindicated by a “*” and generally conserved amino acids are indicated bya “:”.

FIG. 43 depicts a sequence alignment between SEQ ID NO:715 (RAAC04053)and ref|YP_(—)001235093.1|, ref|YP_(—)001547100.1|,ref|ZP_(—)01464237.1|, ref|YP_(—)001509451.1|, and ref|YP_(—)430861.1|(SEQ ID NOS:717-721), respectively, which all have the function assignedto SEQ ID NO:715 in Table 1. Amino acids conserved among all sequencesare indicated by a “*” and generally conserved amino acids are indicatedby a “:”.

FIG. 44 depicts a sequence alignment between SEQ ID NO:732 (RAAC04054)and ref|YP_(—)001547099.1|, gb|AAK01295.1|AF332585_(—)1,ref|YP_(—)001235092.1|, ref|YP_(—)289977.1|, and ref|NP 823422.1|(SEQ IDNOS:734-738), respectively, which all have the function assigned to SEQID NO:732 in Table 1. Amino acids conserved among all sequences areindicated by a “*” and generally conserved amino acids are indicated bya “:”.

FIG. 45 depicts a sequence alignment between SEQ ID NO:749 (RAAC04055)and ref|YP_(—)001547098.1|, ref|ZP_(—)01464218.1|,ref|YP_(—)001509449.1|, ref|YP_(—)001235091.1|, and ref|YP_(—)289976.1|(SEQ ID NOS:751-755), respectively, which all have the function assignedto SEQ ID NO:749 in Table 1. Amino acids conserved among all sequencesare indicated by a “*” and generally conserved amino acids are indicatedby a “:”.

FIGS. 46A and 46B depict a sequence alignment between SEQ ID NO:766(RAAC03004) and ref|YP_(—)001234214.1|, ref|NP_(—)343451.1|,ref|YP_(—)256381.1|, ref|NP 376612.1|, and ref|YP_(—)001191851.1| (SEQID NOS:768-772), respectively, which all have the function assigned toSEQ ID NO:766 in Table 1. Amino acids conserved among all sequences areindicated by a “*” and generally conserved amino acids are indicated bya “:”.

DETAILED DESCRIPTION OF THE INVENTION

Lignocellulose is a highly heterogeneous three-dimensional matrixcomprised primarily of cellulose, hemicellulose, and lignin. Many fuelsand chemicals can be made from these lignocellulosic materials. Toutilize lignocellulosic biomass for production of fuels and chemicalsvia fermentative processes, it is necessary to convert the plantpolysaccharides to sugar monomers, which are then fermented to productsusing a variety of microorganisms. Direct hydrolysis of lignocelluloseby mineral acids to monomers is possible at high temperatures andpressures, leading to yield losses due to thermal decomposition of thesugars. Utilizing existing commercially available enzymes, a firststrategy to reduce these yield losses is to perform the pretreatment ata reduced severity to produce soluble oligomers, followed by the use ofuse cellulases and hemicellulases to depolymerize the polysaccharides atmoderate temperatures. In a second approach, the addition of acid stablethermotolerant hydrolytic enzymes including cellulases, xylanases andother hemicellulases to the biomass slurry during the pretreatmentallows the use of further reduced temperatures and pressures during thepretreatment, as well as cheaper materials of construction, reducingboth the capital and energy costs. An extension of this second approachis to combine the enzyme-assisted reduced severity pretreatment togetherwith fermentation under the same conditions, which further reducescosts.

Regardless of which approach is used, sugars generated by the approachmay be used by cells to create useful fuels and chemicals, such, by wayof non-limiting example, ethanol. Thus, transporters that function inlow pH and/or high temperatures are useful to transport the generatedsugars from the extracellular medium into a cell for secondaryprocessing into a material of interest.

Embodiments of the invention include genes and associated proteinsrelated to the metabolism and sugar transport of the thermoacidophileAlicyclobacillus acidocaldarius. Coding sequences for genes related tothese processes were determined from sequence information generated fromsequencing the genome of Alicyclobacillus acidocaldarius. These genesand proteins may represent targets for metabolic engineering ofAlicyclobacillus acidocaldarius or other organisms. Non-limitingexamples of nucleotide sequences found within the genome ofAlicyclobacillus acidocaldarius, and amino acids coded thereby,associated with sugar transport are listed in Table 1. Sugartransporters and associated molecules may be, without limitation, of thefollowing classes: glucose, galactose, xylose, mannose, arabinose,maltose, lactose, ribose, uronic acids formed from these sugars,acetylated sugars from the aforementioned classes, soluble saccharidesof these sugars including dimers, trimers, and larger oligomers of thesugars both singly and in combination, transporters and associatedmolecules, saccharide transporters and associated molecules;polysaccharide and/or oligosaccharide transporters and associatedmolecules; cyclodextrin, melibiose, cellobiose, galacturonate,oligogalactouronate, glucarate, polyol, chitooligosaccharide,transporters and associated molecules; polyABC-type sugar transportsystems, permease components; Maltose-binding periplasmicproteins/domains; Cyclodextrin-binding proteins;Arabinose/Xylose/Galactose permeases; Sodium-glucose/galactosecotransporters; ABC-type sugar transport systems, ATPase components;Na+/melibiose symporters and related transporters; ABC-type sugartransport system, periplasmic components; Arabinose/Xylose/Galactosepermeases; Sugar binding proteins/transporters; ABC-typepolysaccharide/polyol phosphate export systems, permease components;ABC-type polysaccharide/polyol phosphate transport systems, ATPasecomponents; ABC-type Oligogalacturonate transport system permeaseprotein OgtB; ABC-type Oligogalacturonate transport system permeaseprotein OgtA; ABC-type Oligogalacturonate-binding protein OgtD,periplasmic components; Glucarate/galactarate transporters; Ribosetransport system permease protein rbsC;Ribose/xylose/arabinose/galactoside ABC-type transport systems, permeasecomponents; ABC-type sugar transport system/extracellular sugar bindingproteins, periplasmic components; Cellobiose transporters or regulators;Sugar-binding proteins; Oligosaccharide binding proteins; ABColigosaccharide transporters; ABC oligosaccharidepermeases/transporters; ABC chitooligosaccharide transporters;Ribose/xylose/arabinose/galactoside ABC-type transport systems, permeasecomponents; Ribose/xylose/arabinose/galactoside ABC-type transportsystems, permease components; ABC-type sugar transport system,periplasmic components; Lactose-binding proteins; Lactose transportsystem permease protein lacF; D-ribose-binding proteins;Ribose/xylose/arabinose/galactoside ABC-type transport systems, permeasecomponents; ABC-type xylose transport system, periplasmic components;D-xylose-binding proteins; D-xylose transport ATP-binding protein xylG;ABC-type xylose transport system, permease components; xylH; D-Xyloseproton-symporters; and Sugar kinase activities; and others.

Embodiments of the invention relate in part to the gene sequences and/orprotein sequences comprising genes and/or proteins of Alicyclobacillusacidocaldarius. Genes and proteins included are those that play a rolein sugar transport. Intracellular enzyme activities may be thermophilicand/or acidophilic in nature and general examples of similar genes aredescribed in the literature. Classes of genes, sequences, enzymes andfactors include, but are not limited to, those listed in Table 1.

TABLE 1 Alicyclobacillus acidocaldarius genes and proteins related tosugar transport Protein Reference Sequence Gene Sequence FunctionRAAC00572 SEQ ID NO: 1 SEQ ID NO: 2 ABC-type sugar transport systems,permease components RAAC00573 SEQ ID NO: 18 SEQ ID NO: 19Maltose-binding periplasmic proteins/domains; Cyclodextrin-bindingprotein RAAC00608 SEQ ID NO: 35 SEQ ID NO: 36 Arabinose/Xylose/Galactosepermease RAAC00626 SEQ ID NO: 52 SEQ ID NO: 53 ABC-type sugar transportsystem, periplasmic component RAAC00627 SEQ ID NO: 69 SEQ ID NO: 70ABC-type sugar transport systems, permease components RAAC00628 SEQ IDNO: 86 SEQ ID NO: 87 ABC-type sugar transport system, permease componentRAAC00662 SEQ ID NO: 103 SEQ ID NO: 104 Sodium-glucose/galactosecotransporter RAAC00732 SEQ ID NO: 120 SEQ ID NO: 121 ABC-type sugartransport systems, ATPase components RAAC00804 SEQ ID NO: 137 SEQ ID NO:138 Na+/melibiose symporter and related transporters RAAC00824 SEQ IDNO: 154 SEQ ID NO: 155 ABC-type sugar transport system, periplasmiccomponent RAAC01073 SEQ ID NO: 171 SEQ ID NO: 172Arabinose/Xylose/Galactose permease RAAC01120 SEQ ID NO: 188 SEQ ID NO:189 Sugar binding protein/transport RAAC01122 SEQ ID NO: 205 SEQ ID NO:206 ABC-type sugar transport systems, permease components RAAC01168 SEQID NO: 222 SEQ ID NO: 223 ABC-type polysaccharide/polyol phosphateexport systems, permease component RAAC01169 SEQ ID NO: 239 SEQ ID NO:240 ABC-type polysaccharide/polyol phosphate transport system, ATPasecomponent RAAC01276 SEQ ID NO: 256 SEQ ID NO: 257 ABC-typeOligogalacturonate transport system permease protein OgtB RAAC01277 SEQID NO: 273 SEQ ID NO: 274 ABC-type Oligogalacturonate transport systempermease protein OgtA RAAC01278 SEQ ID NO: 290 SEQ ID NO: 291 ABC-typeOligogalacturonate-binding protein OgtD, periplasmic component RAAC01279SEQ ID NO: 307 SEQ ID NO: 308 ABC-type sugar transport system,periplasmic component RAAC01316 SEQ ID NO: 324 SEQ ID NO: 325Glucarate/galactarate transporter RAAC01502 SEQ ID NO: 341 SEQ ID NO:342 Ribose transport system permease protein rbsC;Ribose/xylose/arabinose/galactoside ABC-type transport systems, permeasecomponents RAAC01599 SEQ ID NO: 358 SEQ ID NO: 359 ABC-type sugartransport systems, permease components RAAC01600 SEQ ID NO: 375 SEQ IDNO: 376 ABC-type sugar transport system, periplasmic component RAAC01625SEQ ID NO: 392 SEQ ID NO: 393 ABC-type sugar transport system, permeasecomponent RAAC01626 SEQ ID NO: 409 SEQ ID NO: 410 ABC-type sugartransport system/extracellular sugar binding protein, periplasmiccomponent RAAC01627 SEQ ID NO: 426 SEQ ID NO: 427 ABC-type sugartransport systems, permease components RAAC01754 SEQ ID NO: 443 SEQ IDNO: 444 Cellobiose transport or regulator RAAC01756 SEQ ID NO: 460 SEQID NO: 461 ABC-type sugar transport system, periplasmic component;Sugar-binding protein RAAC01757 SEQ ID NO: 477 SEQ ID NO: 478 ABC-typesugar transport systems, permease components RAAC01758 SEQ ID NO: 494SEQ ID NO: 495 ABC-type sugar transport system, permease componentRAAC01989 SEQ ID NO: 511 SEQ ID NO: 512 Oligosaccharide binding proteinRAAC01990 SEQ ID NO: 528 SEQ ID NO: 529 ABC oligosaccharide transportRAAC01991 SEQ ID NO: 545 SEQ ID NO: 546 ABC oligosaccharidepermease/transport RAAC01992 SEQ ID NO: 562 SEQ ID NO: 563 ABCchitooligosaccharide transport RAAC02175 SEQ ID NO: 579 SEQ ID NO: 580ABC-type sugar transport system, periplasmic component RAAC02176 SEQ IDNO: 596 SEQ ID NO: 597 Ribose/xylose/arabinose/galactoside ABC-typetransport systems, permease components RAAC02177 SEQ ID NO: 613 SEQ IDNO: 614 Ribose/xylose/arabinose/galactoside ABC-type transport systems,permease components RAAC02613 SEQ ID NO: 630 SEQ ID NO: 631 ABC-typesugar transport system, periplasmic component; Lactose-binding proteinRAAC02614 SEQ ID NO: 647 SEQ ID NO: 648 ABC-type sugar transportsystems, permease components; Lactose transport system permease proteinlacF RAAC02615 SEQ ID NO: 664 SEQ ID NO: 665 ABC-type sugar transportsystem, permease component RAAC02733 SEQ ID NO: 681 SEQ ID NO: 682ABC-type sugar transport system, periplasmic component; D-ribose-bindingprotein RAAC02734 SEQ ID NO: 698 SEQ ID NO: 699Ribose/xylose/arabinose/galactoside ABC-type transport systems, permeasecomponents RAAC04053 SEQ ID NO: 715 SEQ ID NO: 716 ABC-type xylosetransport system, periplasmic component; D-xylose-binding proteinRAAC04054 SEQ ID NO: 732 SEQ ID NO: 733 D-xylose transport ATP-bindingprotein xylG RAAC04055 SEQ ID NO: 749 SEQ ID NO: 750 ABC-type xylosetransport system, permease component; xylH RAAC03004 SEQ ID NO: 766 SEQID NO: 767 D-XYLOSE PROTON-SYMPORTER; Predicted sugar kinase

The present invention relates to nucleotides sequences comprisingisolated and/or purified nucleotide sequences of the genome ofAlicyclobacillus acidocaldarius selected from the sequences of SEQ IDNOS:2, 19, 36, 53, 70, 87, 104, 121, 138, 155, 172, 189, 206, 223, 240,257, 274, 291, 308, 325, 342, 359, 376, 393, 410, 427, 444, 461, 478,495, 512, 529, 546, 563, 580, 597, 614, 631, 648, 665, 682, 699, 716,733, 750, and 767, or one of their fragments.

The present invention likewise relates to isolated and/or purifiednucleotide sequences, characterized in that they comprise at least oneof: a) a nucleotide sequence of at least one of the sequences of SEQ IDNOS:2, 19, 36, 53, 70, 87, 104, 121, 138, 155, 172, 189, 206, 223, 240,257, 274, 291, 308, 325, 342, 359, 376, 393, 410, 427, 444, 461, 478,495, 512, 529, 546, 563, 580, 597, 614, 631, 648, 665, 682, 699, 716,733, 750, and 767 or one of their fragments; b) a nucleotide sequencehomologous to a nucleotide sequence such as defined in a); c) anucleotide sequence complementary to a nucleotide sequence such asdefined in a) or b), and a nucleotide sequence of their correspondingRNA; d) a nucleotide sequence capable of hybridizing under stringentconditions with a sequence such as defined in a), b) or c); e) anucleotide sequence comprising a sequence such as defined in a), b), c)or d); and f) a nucleotide sequence modified by a nucleotide sequencesuch as defined in a), b), c), d) or e).

Nucleotide, polynucleotide, or nucleic acid sequence will be understoodaccording to the present invention as meaning both a double-stranded orsingle-stranded DNA in the monomeric and dimeric (so-called in tandem)forms and the transcription products of the DNAs.

Aspects of the invention relate nucleotide sequences, which it has beenpossible to isolate, purify or partially purify, starting fromseparation methods such as, for example, ion-exchange chromatography, byexclusion based on molecular size, or by affinity, or alternativelyfractionation techniques based on solubility in different solvents, orstarting from methods of genetic engineering such as amplification,cloning, and subcloning, it being possible for the sequences of theinvention to be carried by vectors.

Isolated and/or purified nucleotide sequence fragment according to theinvention will be understood as designating any nucleotide fragment ofthe genome of Alicyclobacillus acidocaldarius, and may include, by wayof non-limiting examples, length of at least 8, 12, 20 25, 50, 75, 100,200, 300, 400, 500, 1000, or more, consecutive nucleotides of thesequence from which it originates.

Specific fragment of an isolated and/or purified nucleotide sequenceaccording to the invention will be understood as designating anynucleotide fragment of the genome of Alicyclobacillus acidocaldarius,having, after alignment and comparison with the corresponding fragmentsof genomic sequences of Alicyclobacillus acidocaldarius, at least onenucleotide or base of different nature.

Homologous isolated and/or purified nucleotide sequence, in the sense ofthe present invention, is understood as meaning isolated and/or purifieda nucleotide sequence having at least a percentage identity with thebases of a nucleotide sequence according to the invention of at leastabout 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%,93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.6%, or 99.7%, thispercentage being purely statistical and it being possible to distributethe differences between the two nucleotide sequences at random and overthe whole of their length.

Specific homologous nucleotide sequence in the sense of the presentinvention is understood as meaning a homologous nucleotide sequencehaving at least one nucleotide sequence of a specific fragment, such asdefined above. The “specific” homologous sequences can comprise, forexample, the sequences corresponding to the genomic sequence or to thesequences of its fragments representative of variants of the genome ofAlicyclobacillus acidocaldarius. These specific homologous sequences canthus correspond to variations linked to mutations within strains ofAlicyclobacillus acidocaldarius, and especially correspond totruncations, substitutions, deletions and/or additions of at least onenucleotide. The homologous sequences can likewise correspond tovariations linked to the degeneracy of the genetic code.

The term “degree or percentage of sequence homology” refers to “degreeor percentage of sequence identity between two sequences after optimalalignment” as defined in the present application.

Two amino-acids or nucleotidic sequences are said to be “identical” ifthe sequence of amino-acids or nucleotidic residues, in the twosequences is the same when aligned for maximum correspondence asdescribed below. Sequence comparisons between two (or more) peptides orpolynucleotides are typically performed by comparing sequences of twooptimally aligned sequences over a segment or “comparison window” toidentify and compare local regions of sequence similarity. Optimalalignment of sequences for comparison may be conducted by the localhomology algorithm of Smith and Waterman, Ad. App. Math 2:482 (1981), bythe homology alignment algorithm of Neddleman and Wunsch, J. Mol. Biol.48:443 (1970), by the search for similarity method of Pearson andLipman, Proc. Natl. Acad. Sci. (U.S.A.) 85:2444 (1988), by computerizedimplementation of these algorithms (GAP, BESTFIT, FASTA, and TFASTA inthe Wisconsin Genetics Software Package, Genetics Computer Group (GCG),575 Science Dr., Madison, Wis.), or by visual inspection.

“Percentage of sequence identity” (or degree of identity) is determinedby comparing two optimally aligned sequences over a comparison window,where the portion of the peptide or polynucleotide sequence in thecomparison window may comprise additions or deletions (i.e., gaps) ascompared to the reference sequence (which does not comprise additions ordeletions) for optimal alignment of the two sequences. The percentage iscalculated by determining the number of positions at which the identicalamino-acid residue or nucleic acid base occurs in both sequences toyield the number of matched positions, dividing the number of matchedpositions by the total number of positions in the window of comparisonand multiplying the result by 100 to yield the percentage of sequenceidentity.

The definition of “sequence identity,” given above, is the definitionthat would be used by one of skill in the art. The definition by itselfdoes not need the help of any algorithm, the algorithms being helpfulonly to achieve the optimal alignments of sequences, rather than thecalculation of sequence identity.

From the definition given above, it follows that there is a well-definedand only one value for the sequence identity between two comparedsequences which value corresponds to the value obtained for the best oroptimal alignment.

In the BLAST N or BLAST P “BLAST 2 sequence” software, which isavailable on the Internet from the web sitencbi.nlm.nih.gov/gorf/bl2.html, and habitually used by the inventors andin general by the skilled person for comparing and determining theidentity between two sequences, gap cost that depends on the sequencelength to be compared is directly selected by the software (i.e., 11.2for substitution matrix BLOSUM-62 for length>85).

Complementary nucleotide sequence of a sequence of the invention isunderstood as meaning any DNA whose nucleotides are complementary tothose of the sequence of the invention, and whose orientation isreversed (antisense sequence).

Hybridization under conditions of stringency with a nucleotide sequenceaccording to the invention is understood as meaning hybridization underconditions of temperature and ionic strength chosen in such a way thatthey allow the maintenance of the hybridization between two fragments ofcomplementary DNA.

By way of illustration, conditions of great stringency of thehybridization step with the aim of defining the nucleotide fragmentsdescribed above are advantageously the following.

The hybridization is carried out at a preferential temperature of 65° C.in the presence of SSC buffer, 1×SSC corresponding to 0.15 M NaCl and0.05 M Na citrate. The washing steps, for example, can be the following:2×SSC, at ambient temperature followed by two washes with 2×SSC, 0.5%SDS at 65° C.; 2×0.5×SSC, 0.5% SDS; at 65° C. for 10 minutes each.

The conditions of intermediate stringency, using, for example, atemperature of 42° C. in the presence of a 2×SSC buffer, or of lessstringency, for example, a temperature of 37° C. in the presence of a2×SSC buffer, respectively require a globally less significantcomplementarity for the hybridization between the two sequences.

The stringent hybridization conditions described above for apolynucleotide with a size of approximately 350 bases will be adapted bya person skilled in the art for oligonucleotides of greater or smallersize, according to the teaching of Sambrook et al., 1989.

Among the isolated and/or purified nucleotide sequences according to theinvention, are those that can be used as a primer or probe in methodsallowing the homologous sequences according to the invention to beobtained, these methods, such as the polymerase chain reaction (PCR),nucleic acid cloning, and sequencing, being well known to the personskilled in the art.

Among the isolated and/or purified nucleotide sequences according to theinvention, those are again preferred that can be used as a primer orprobe in methods allowing the presence of SEQ ID NOS:2, 19, 36, 53, 70,87, 104, 121, 138, 155, 172, 189, 206, 223, 240, 257, 274, 291, 308,325, 342, 359, 376, 393, 410, 427, 444, 461, 478, 495, 512, 529, 546,563, 580, 597, 614, 631, 648, 665, 682, 699, 716, 733, 750, and 767, oneof their fragments, or one of their variants such as defined below to bediagnosed.

The nucleotide sequence fragments according to the invention can beobtained, for example, by specific amplification, such as PCR, or afterdigestion with appropriate restriction enzymes of nucleotide sequencesaccording to the invention, these methods in particular being describedin the work of Sambrook et al., 1989. Such representative fragments canlikewise be obtained by chemical synthesis according to methods wellknown to persons of ordinary skill in the art.

“Modified nucleotide sequence” will be understood as meaning anynucleotide sequence obtained by mutagenesis according to techniques wellknown to the person skilled in the art, and containing modificationswith respect to the normal sequences according to the invention, forexample mutations in the regulatory and/or promoter sequences ofpolypeptide expression, especially leading to a modification of the rateof expression of the polypeptide or to a modulation of the replicativecycle.

“Modified nucleotide sequence” will likewise be understood as meaningany nucleotide sequence coding for a modified polypeptide such asdefined below.

The present invention relates to nucleotide sequence comprising isolatedand/or purified nucleotide sequences of Alicyclobacillus acidocaldarius,characterized in that they are selected from the sequences SEQ ID NOS:2,19, 36, 53, 70, 87, 104, 121, 138, 155, 172, 189, 206, 223, 240, 257,274, 291, 308, 325, 342, 359, 376, 393, 410, 427, 444, 461, 478, 495,512, 529, 546, 563, 580, 597, 614, 631, 648, 665, 682, 699, 716, 733,750, and 767 or one of their fragments.

Embodiments of the invention likewise relate to isolated and/or purifiednucleotide sequences characterized in that they comprise a nucleotidesequence selected from: a) at least one of a nucleotide sequence of SEQID NOS:2, 19, 36, 53, 70, 87, 104, 121, 138, 155, 172, 189, 206, 223,240, 257, 274, 291, 308, 325, 342, 359, 376, 393, 410, 427, 444, 461,478, 495, 512, 529, 546, 563, 580, 597, 614, 631, 648, 665, 682, 699,716, 733, 750, and 767 or one of their fragments or one of theirfragments; b) a nucleotide sequence of a specific fragment of a sequencesuch as defined in a); c) a homologous nucleotide sequence having atleast 80% identity with a sequence such as defined in a) or b); d) acomplementary nucleotide sequence or sequence of RNA corresponding to asequence such as defined in a), b) or c); and e) a nucleotide sequencemodified by a sequence such as defined in a), b), c) or d).

Among the isolated and/or purified nucleotide sequences according to theinvention are the nucleotide sequences of SEQ ID NOS:13-17, 30-34,47-51, 64-68, 81-85, 98-102, 115-119, 132-136, 149-153, 166-170,183-187, 200-204, 217-221, 234-238, 251-255, 268-272, 285-289, 302-306,319-323, 336-340, 353-357, 370-374, 387-391, 404-408, 421-425, 438-442,455-459, 472-476, 489-493, 506-510, 523-527, 540-544, 557-561, 574-578,591-595, 608-612, 625-629, 642-646, 659-663, 676-680, 693-697, 710-714,727-731, 744-748, 761-765, and 778-782, or fragments thereof and anyisolated and/or purified nucleotide sequences, which have a homology ofat least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%,92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.6%, or 99.7% identitywith the at least one of the sequences of SEQ ID NOS:2, 19, 36, 53, 70,87, 104, 121, 138, 155, 172, 189, 206, 223, 240, 257, 274, 291, 308,325, 342, 359, 376, 393, 410, 427, 444, 461, 478, 495, 512, 529, 546,563, 580, 597, 614, 631, 648, 665, 682, 699, 716, 733, 750, and 767 orfragments thereof. The homologous sequences can comprise, for example,the sequences corresponding to the genomic sequences Alicyclobacillusacidocaldarius. In the same manner, these specific homologous sequencescan correspond to variations linked to mutations within strains ofAlicyclobacillus acidocaldarius and especially correspond totruncations, substitutions, deletions and/or additions of at least onenucleotide. As will be apparent to one of ordinary skill in the art,such homologues are easily created and identified using standardtechniques and publicly available computer programs such as BLAST. Assuch, each homologue referenced above should be considered as set forthherein and fully described.

Embodiments of the invention comprise the isolated and/or purifiedpolypeptides coded for by a nucleotide sequence according to theinvention, or fragments thereof, whose sequence is represented by afragment. Amino acid sequences corresponding to the isolated and/orpurified polypeptides, which can be coded for according to one of thethree possible reading frames of at least one of the sequences of SEQ IDNOS:2, 19, 36, 53, 70, 87, 104, 121, 138, 155, 172, 189, 206, 223, 240,257, 274, 291, 308, 325, 342, 359, 376, 393, 410, 427, 444, 461, 478,495, 512, 529, 546, 563, 580, 597, 614, 631, 648, 665, 682, 699, 716,733, 750, and 767.

Embodiments of the invention likewise relate to the isolated and/orpurified polypeptides, characterized in that they comprise a polypeptideselected from at least one of the amino acid sequences of SEQ ID NOS:1,18, 35, 52, 69, 86, 103, 120, 137, 154, 171, 188, 205, 222, 239, 256,273, 290, 307, 324, 341, 358, 375, 392, 409, 426, 443, 460, 477, 494,511, 528, 545, 562, 579, 596, 613, 630, 647, 664, 681, 698, 715, 732,749, and 766 or one of their fragments.

Among the isolated and/or purified polypeptides, according toembodiments of the invention, are the isolated and/or purifiedpolypeptides of amino acid sequence SEQ ID NOS:8-12, 25-29, 42-46,59-63, 76-80, 93-97, 110-114, 127-131, 144-148, 161-165, 178-182,195-199, 212-216, 229-233, 246-250, 263-267, 280-284, 297-301, 314-318,331-335, 348-352, 365-369, 382-386, 399-403, 416-420, 433-437, 450-454,467-471, 484-488, 501-505, 518-522, 535-539, 552-556, 569-573, 586-590,603-607, 620-624, 637-641, 654-658, 671-675, 688-692, 705-709, 722-726,739-743, 756-760, and 773-777, or fragments thereof or any otherisolated and/or purified polypeptides that have a homology of at least80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%,94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.6%, or 99.7% identity with atleast one of the sequences of SEQ ID NOS:1, 18, 35, 52, 69, 86, 103,120, 137, 154, 171, 188, 205, 222, 239, 256, 273, 290, 307, 324, 341,358, 375, 392, 409, 426, 443, 460, 477, 494, 511, 528, 545, 562, 579,596, 613, 630, 647, 664, 681, 698, 715, 732, 749, and 766 or fragmentsthereof. As will be apparent to one of ordinary skill in the art, suchhomologues are easily created and identified using standard techniquesand publicly available computer programs such as BLAST. As such, eachhomologue referenced above should be considered as set forth herein andfully described.

Embodiments of the invention also relate to the polypeptides,characterized in that they comprise a polypeptide selected from: a) aspecific fragment of at least 5 amino acids of a polypeptide of an aminoacid sequence according to the invention; b) a polypeptide homologous toa polypeptide such as defined in a); c) a specific biologically activefragment of a polypeptide such as defined in a) or b); and d) apolypeptide modified by a polypeptide such as defined in a), b) or c).

In the present description, the terms polypeptide, peptide and proteinare interchangeable.

In embodiments of the invention, the isolated and/or purifiedpolypeptides according to the invention may be glycosylated, pegylated,and/or otherwise post-translationally modified. In further embodiments,glycosylation, pegylation, and/or other post-translational modificationsmay occur in vivo or in vitro and/or may be performed using chemicaltechniques. In additional embodiments, any glycosylation, pegylationand/or other post-translational modifications may be N-linked orO-linked.

In embodiments of the invention, any one of the isolated and/or purifiedpolypeptides according to the invention may be enzymatically orfunctionally active at temperatures at or above about 25, 30, 35, 40,45, 50, 55, 60, 65, 70, 75, 80, 85, 90, and/or 95 degrees Celsius and/ormay be enzymatically or functionally active at a pH at, below, and/orabove 8, 7, 6, 5, 4, 3, 2, 1, and/or 0. In further embodiments of theinvention, glycosylation, pegylation, and/or other post-translationalmodification may be required for the isolated and/or purifiedpolypeptides according to the invention to be enzymatically orfunctionally active at a pH at or below 8, 7, 6, 5, 4, 3, 2, 1, and/or 0or at temperatures at or above about 25, 30, 35, 40, 45, 50, 55, 60, 65,70, 75, 80, 85, 90, and/or 95 degrees Celsius.

Aspects of the invention relate to polypeptides that are isolated orobtained by purification from natural sources, or else obtained bygenetic recombination, or alternatively by chemical synthesis and thatthey may thus contain unnatural amino acids, as will be described below.

A “polypeptide fragment” according to the embodiments of the inventionis understood as designating a polypeptide containing at least five (5)consecutive amino acids, preferably ten (10) consecutive amino acids orfifteen (15) consecutive amino acids.

In the present invention, a specific polypeptide fragment is understoodas designating the consecutive polypeptide fragment coded for by aspecific fragment nucleotide sequence according to the invention.

“Homologous polypeptide” will be understood as designating thepolypeptides having, with respect to the natural polypeptide, certainmodifications such as, in particular, a deletion, addition, orsubstitution of at least one amino acid, a truncation, a prolongation, achimeric fusion, and/or a mutation. Among the homologous polypeptides,those are preferred whose amino acid sequence has at least 80% or 90%,homology with the sequences of amino acids of polypeptides according tothe invention.

“Specific homologous polypeptide” will be understood as designating thehomologous polypeptides such as defined above and having a specificfragment of polypeptide according to the invention.

In the case of a substitution, one or more consecutive or nonconsecutiveamino acids are replaced by “equivalent” amino acids. The expression“equivalent” amino acid is directed here at designating any amino acidcapable of being substituted by one of the amino acids of the basestructure without, however, essentially modifying the biologicalactivities of the corresponding peptides and such that they will bedefined by the following. As will be apparent to one of ordinary skillin the art, such substitutions are easily created and identified usingstandard molecular biology techniques and publicly available computerprograms such as BLAST. As such, each substitution referenced aboveshould be considered as set forth herein and fully described. Examplesof such substitutions in the amino acid sequences SEQ ID NOS:1, 18, 35,52, 69, 86, 103, 120, 137, 154, 171, 188, 205, 222, 239, 256, 273, 290,307, 324, 341, 358, 375, 392, 409, 426, 443, 460, 477, 494, 511, 528,545, 562, 579, 596, 613, 630, 647, 664, 681, 698, 715, 732, 749, and 766may include those isolated and/or purified polypeptides of amino acidsequence SEQ ID NOS:8-12, 25-29, 42-46, 59-63, 76-80, 93-97, 110-114,127-131, 144-148, 161-165, 178-182, 195-199, 212-216, 229-233, 246-250,263-267, 280-284, 297-301, 314-318, 331-335, 348-352, 365-369, 382-386,399-403, 416-420, 433-437, 450-454, 467-471, 484-488, 501-505, 518-522,535-539, 552-556, 569-573, 586-590, 603-607, 620-624, 637-641, 654-658,671-675, 688-692, 705-709, 722-726, 739-743, 756-760, and 773-777. Theseequivalent amino acids may be determined either by depending on theirstructural homology with the amino acids that they substitute, or onresults of comparative tests of biological activity between thedifferent polypeptides, which are capable of being carried out.

By way of non-limiting example, the possibilities of substitutionscapable of being carried out without resulting in an extensivemodification of the biological activity of the corresponding modifiedpolypeptides will be mentioned, the replacement, for example, of leucineby valine or isoleucine, of aspartic acid by glutamic acid, of glutamineby asparagine, of arginine by lysine etc., the reverse substitutionsnaturally being envisageable under the same conditions.

In a further embodiment, substitutions are limited to substitutions inamino acids not conserved among other proteins that have similaridentified enzymatic activity. For example, one of ordinary skill in theart may align proteins of the same function in similar organisms anddetermine which amino acids are generally conserved among proteins ofthat function. One example of a program that may be used to generatesuch alignments is available on the worldwide web atcharite.de/bioinf/strap/ in conjunction with the databases provided bythe NCBI. Examples of such polypeptides may include, but are not limitedto, those found in amino acid sequence SEQ ID NOS:8-12, 25-29, 42-46,59-63, 76-80, 93-97, 110-114, 127-131, 144-148, 161-165, 178-182,195-199, 212-216, 229-233, 246-250, 263-267, 280-284, 297-301, 314-318,331-335, 348-352, 365-369, 382-386, 399-403, 416-420, 433-437, 450-454,467-471, 484-488, 501-505, 518-522, 535-539, 552-556, 569-573, 586-590,603-607, 620-624, 637-641, 654-658, 671-675, 688-692, 705-709, 722-726,739-743, 756-760, and 773-777.

Thus, according to one embodiment of the invention, substitutions ormutation may be made at positions that are generally conserved amongproteins of that function. In a further embodiment, nucleic acidsequences may be mutated or substituted such that the amino acid theycode for is unchanged (degenerate substitutions and/mutations) and/ormutated or substituted such that any resulting amino acid substitutionsor mutation are made at positions that are generally conserved amongproteins of that function. Examples of such nucleic acid sequences mayinclude, but are not limited to, those found in are the nucleotidesequences of SEQ ID NOS:13-17, 30-34, 47-51, 64-68, 81-85, 98-102,115-119, 132-136, 149-153, 166-170, 183-187, 200-204, 217-221, 234-238,251-255, 268-272, 285-289, 302-306, 319-323, 336-340, 353-357, 370-374,387-391, 404-408, 421-425, 438-442, 455-459, 472-476, 489-493, 506-510,523-527, 540-544, 557-561, 574-578, 591-595, 608-612, 625-629, 642-646,659-663, 676-680, 693-697, 710-714, 727-731, 744-748, 761-765, 778-782or fragments thereof.

The specific homologous polypeptides likewise correspond to polypeptidescoded for by the specific homologous nucleotide sequences such asdefined above and thus comprise in the present definition thepolypeptides, which are mutated or correspond to variants, which canexist in Alicyclobacillus acidocaldarius, and which especiallycorrespond to truncations, substitutions, deletions, and/or additions ofat least one amino acid residue.

“Specific biologically active fragment of a polypeptide” according to anembodiment of the invention will be understood in particular asdesignating a specific polypeptide fragment, such as defined above,having at least one of the characteristics of polypeptides according tothe invention. In certain embodiments the peptide is capable of behavingas at least one of the types of proteins outlined in Table 1.

The polypeptide fragments according to embodiments of the invention cancorrespond to isolated or purified fragments naturally present inAlicyclobacillus acidocaldarius or correspond to fragments that can beobtained by cleavage of the polypeptide by a proteolytic enzyme, such astrypsin or chymotrypsin or collagenase, or by a chemical reagent, suchas cyanogen bromide (CNBr). Such polypeptide fragments can likewise justas easily be prepared by chemical synthesis, from hosts transformed byan expression vector according to the invention containing a nucleicacid allowing the expression of the fragments, placed under the controlof appropriate regulation and/or expression elements.

“Modified polypeptide” of a polypeptide according to an embodiment ofthe invention is understood as designating a polypeptide obtained bygenetic recombination or by chemical synthesis as will be describedbelow, having at least one modification with respect to the normalsequence. These modifications may or may not be able to bear on aminoacids at the origin of specificity, and/or of activity, or at the originof the structural conformation, localization, and of the capacity ofmembrane insertion of the polypeptide according to the invention. Itwill thus be possible to create polypeptides of equivalent, increased,or decreased activity, and of equivalent, narrower, or widerspecificity. Among the modified polypeptides, it is necessary to mentionthe polypeptides in which up to five (5) or more amino acids can bemodified, truncated at the N- or C-terminal end, or even deleted oradded.

The methods allowing the modulations on eukaryotic or prokaryotic cellsto be demonstrated are well known to the person of ordinary skill in theart. It is likewise well understood that it will be possible to use thenucleotide sequences coding for the modified polypeptides for themodulations, for example, through vectors according to the invention anddescribed below.

The preceding modified polypeptides can be obtained by usingcombinatorial chemistry, in which it is possible to systematically varyparts of the polypeptide before testing them on models, cell cultures ormicroorganisms, for example, to select the compounds that are mostactive or have the properties sought.

Chemical synthesis likewise has the advantage of being able to useunnatural amino acids, or nonpeptide bonds.

Thus, in order to improve the duration of life of the polypeptidesaccording to the invention, it may be of interest to use unnatural aminoacids, for example in D faun, or else amino acid analogs, especiallysulfur-containing forms, for example.

Finally, it will be possible to integrate the structure of thepolypeptides according to the invention, its specific or modifiedhomologous forms, into chemical structures of polypeptide type orothers. Thus, it may be of interest to provide at the N- and C-terminalends molecules not recognized by proteases.

The nucleotide sequences coding for a polypeptide according to theinvention are likewise part of the invention.

The invention likewise relates to nucleotide sequences utilizable as aprimer or probe, characterized in that the sequences are selected fromthe nucleotide sequences according to the invention.

It is well understood that the present invention, in variousembodiments, likewise relates to specific polypeptides ofAlicyclobacillus acidocaldarius, coded for by nucleotide sequences,capable of being obtained by purification from natural polypeptides, bygenetic recombination or by chemical synthesis by procedures well knownto a person skilled in the art and such as described in particularbelow. In the same manner, the labeled or unlabeled mono- or polyclonalantibodies directed against the specific polypeptides coded for by thenucleotide sequences are also encompassed by the invention.

Embodiments of the invention additionally relate to the use of anucleotide sequence according to the invention as a primer or probe forthe detection and/or the amplification of nucleic acid sequences.

The nucleotide sequences according to embodiments of the invention canthus be used to amplify nucleotide sequences, especially by the PCR(polymerase chain reaction) technique (Erlich, 1989; Innis et al., 1990;Rolfs et al., 1991; and White et al., 1997).

These oligodeoxyribonucleotide or oligoribonucleotide primersadvantageously have a length of at least eight (8) nucleotides,preferably of at least twelve (12) nucleotides, and even morepreferentially at least twenty (20) nucleotides.

Other amplification techniques of the target nucleic acid can beadvantageously employed as alternatives to PCR.

The nucleotide sequences of the invention, in particular the primersaccording to the invention, can likewise be employed in other proceduresof amplification of a target nucleic acid, such as: the TAS(Transcription-based Amplification System) technique, described by Kwohet al. in 1989; the 3SR (Self-Sustained Sequence Replication) technique,described by Guatelli et al. in 1990; the NASBA (Nucleic Acid SequenceBased Amplification) technique, described by Kievits et al. in 1991; theSDA (Strand Displacement Amplification) technique (Walker et al., 1992);the TMA (Transcription Mediated Amplification) technique.

The polynucleotides of the invention can also be employed in techniquesof amplification or of modification of the nucleic acid serving as aprobe, such as: the LCR (Ligase Chain Reaction) technique, described byLandegren et al. in 1988 and improved by Barany et al. in 1991, whichemploys a thermostable ligase; the RCR (Repair Chain Reaction)technique, described by Segev in 1992; the CPR (Cycling Probe Reaction)technique, described by Duck et al. in 1990; the amplification techniquewith Q-beta replicase, described by Miele et al. in 1983 and especiallyimproved by Chu et al. in 1986, Lizardi et al. in 1988, then by Burg etal., as well as by Stone et al. in 1996.

In the case where the target polynucleotide to be detected is possiblyan RNA, for example an mRNA, it will be possible to use, prior to theemployment of an amplification reaction with the aid of at least oneprimer according to the invention or to the employment of a detectionprocedure with the aid of at least one probe of the invention, an enzymeof reverse transcriptase type in order to obtain a cDNA from the RNAcontained in the biological sample. The cDNA obtained will thus serve asa target for the primer(s) or the probe(s) employed in the amplificationor detection procedure according to the invention.

The detection probe will be chosen in such a manner that it hybridizeswith the target sequence or the amplicon generated from the targetsequence. By way of sequence, such a probe will advantageously have asequence of at least twelve (12) nucleotides, in particular of at leasttwenty (20) nucleotides, and preferably of at least one hundred (100)nucleotides.

Embodiments of the invention also comprise the nucleotide sequencesutilizable as a probe or primer according to the invention,characterized in that they are labeled with a radioactive compound orwith a nonradioactive compound.

The unlabeled nucleotide sequences can be used directly as probes orprimers, although the sequences are generally labeled with a radioactiveisotope (³²P_(,) ³⁵S, ³H, ¹²⁵I) or with a nonradioactive molecule(biotin, acetylaminofluorene, digoxigenin, 5-bromodeoxyuridine,fluorescein) to obtain probes that are utilizable for numerousapplications.

Examples of nonradioactive labeling of nucleotide sequences aredescribed, for example, in French Patent No. 7810975 or by Urdea et al.or by Sanchez-Pescador et al., both in 1988.

In the latter case, it will also be possible to use one of the labelingmethods described in patents FR-2 422 956 and FR-2 518 755.

The hybridization technique can be carried out in various manners(Matthews et al., 1988). The most general method consists inimmobilizing the nucleic acid extract of cells on a support (such asnitrocellulose, nylon, polystyrene) and in incubating, underwell-defined conditions, the immobilized target nucleic acid with theprobe. After hybridization, the excess of probe is eliminated and thehybrid molecules formed are detected by the appropriate method(measurement of the radioactivity, of the fluorescence or of theenzymatic activity linked to the probe).

The invention, in various embodiments, likewise comprises the nucleotidesequences according to the invention, characterized in that they areimmobilized on a support, covalently or noncovalently.

According to another advantageous mode of employing nucleotide sequencesaccording to the invention, the latter can be used immobilized on asupport and can thus serve to capture, by specific hybridization, thetarget nucleic acid obtained from the biological sample to be tested. Ifnecessary, the solid support is separated from the sample and thehybridization complex formed between the capture probe, and the targetnucleic acid is then detected with the aid of a second probe, aso-called detection probe, labeled with an easily detectable element.

Another aspect of the present invention is a vector for the cloningand/or expression of a sequence, characterized in that it contains anucleotide sequence according to the invention.

The vectors according to the invention, characterized in that theycontain the elements allowing the integration, expression and/or thesecretion of the nucleotide sequences in a determined host cell, arelikewise part of the invention.

The vector may then contain a promoter, signals of initiation andtermination of translation, as well as appropriate regions of regulationof transcription. It may be able to be maintained stably in the hostcell and can optionally have particular signals specifying the secretionof the translated protein. These different elements may be chosen as afunction of the host cell used. To this end, the nucleotide sequencesaccording to the invention may be inserted into autonomous replicationvectors within the chosen host, or integrated vectors of the chosenhost.

Such vectors will be prepared according to the methods currently used bythe person skilled in the art, and it will be possible to introduce theclones resulting therefrom into an appropriate host by standard methods,such as, for example, lipofection, electroporation, and thermal shock.

The vectors according to the invention are, for example, vectors ofplasmid or viral origin. One example of a vector for the expression ofpolypeptides of the invention is baculovirus.

These vectors are useful for transforming host cells in order to cloneor to express the nucleotide sequences of the invention.

The invention likewise comprises the host cells transformed by a vectoraccording to the invention.

These cells can be obtained by the introduction into host cells of anucleotide sequence inserted into a vector such as defined above, thenthe culturing of the cells under conditions allowing the replicationand/or expression of the transfected nucleotide sequence.

The host cell can be selected from prokaryotic or eukaryotic systems,such as, for example, bacterial cells (Olins and Lee, 1993), butlikewise yeast cells (Buckholz, 1993), as well as plants cells, such asArabidopsis sp., and animal cells, in particular the cultures ofmammalian cells (Edwards and Aruffo, 1993), for example, Chinese hamsterovary (CHO) cells, but likewise the cells of insects in which it ispossible to use procedures employing baculoviruses, for example sf9insect cells (Luckow, 1993).

Embodiments of the invention likewise relate to organisms comprising oneof the transformed cells according to the invention.

The obtainment of transgenic organisms according to the inventionexpressing one or more of the genes of Alicyclobacillus acidocaldariusor part of the genes may be carried out in, for example, rats, mice, orrabbits according to methods well known to the person skilled in theart, such as by viral or nonviral transfections. It will be possible toobtain the transgenic organisms expressing one or more of the genes bytransfection of multiple copies of the genes under the control of astrong promoter of ubiquitous nature, or selective for one type oftissue. It will likewise be possible to obtain the transgenic organismsby homologous recombination in embryonic cell strains, transfer of thesecell strains to embryos, selection of the affected chimeras at the levelof the reproductive lines, and growth of the chimeras.

The transformed cells as well as the transgenic organisms according tothe invention are utilizable in procedures for preparation ofrecombinant polypeptides.

It is today possible to produce recombinant polypeptides in relativelylarge quantity by genetic engineering using the cells transformed byexpression vectors according to the invention or using transgenicorganisms according to the invention.

The procedures for preparation of a polypeptide of the invention inrecombinant form, characterized in that they employ a vector and/or acell transformed by a vector according to the invention and/or atransgenic organism comprising one of the transformed cells according tothe invention are themselves comprised in the present invention.

As used herein, “transformation” and “transformed” relate to theintroduction of nucleic acids into a cell, whether prokaryotic oreukaryotic. Further, “transformation” and “transformed,” as used herein,need not relate to growth control or growth deregulation.

Among the procedures for preparation of a polypeptide of the inventionin recombinant form, the preparation procedures employing a vector,and/or a cell transformed by the vector and/or a transgenic organismcomprising one of the transformed cells, containing a nucleotidesequence according to the invention coding for a polypeptide ofAlicyclobacillus acidocaldarius.

A variant, according to the invention, may consist of producing arecombinant polypeptide fused to a “carrier” protein (chimeric protein).The advantage of this system is that it may allow stabilization ofand/or a decrease in the proteolysis of the recombinant product, anincrease in the solubility in the course of renaturation in vitro and/ora simplification of the purification when the fusion partner has anaffinity for a specific ligand.

More particularly, the invention relates to a procedure for preparationof a polypeptide of the invention comprising the following steps: a)culture of transformed cells under conditions allowing the expression ofa recombinant polypeptide of nucleotide sequence according to theinvention; b) if need be, recovery of the recombinant polypeptide.

When the procedure for preparation of a polypeptide of the inventionemploys a transgenic organism according to the invention, therecombinant polypeptide is then extracted from the organism.

The invention also relates to a polypeptide that is capable of beingobtained by a procedure of the invention such as described previously.

The invention also comprises a procedure for preparation of a syntheticpolypeptide, characterized in that it uses a sequence of amino acids ofpolypeptides according to the invention.

The invention likewise relates to a synthetic polypeptide obtained by aprocedure according to the invention.

The polypeptides according to the invention can likewise be prepared bytechniques that are conventional in the field of the synthesis ofpeptides. This synthesis can be carried out in homogeneous solution orin solid phase.

For example, recourse can be made to the technique of synthesis inhomogeneous solution described by Houben-Weyl in 1974.

This method of synthesis consists in successively condensing, two bytwo, the successive amino acids in the order required, or in condensingamino acids and fragments formed previously and already containingseveral amino acids in the appropriate order, or alternatively severalfragments previously prepared in this way, it being understood that itwill be necessary to protect beforehand all the reactive functionscarried by these amino acids or fragments, with the exception of aminefunctions of one and carboxyls of the other or vice versa, which mustnormally be involved in the formation of peptide bonds, especially afteractivation of the carboxyl function, according to the methods well knownin the synthesis of peptides.

Recourse may also be made to the technique described by Merrifield.

To make a peptide chain according to the Merrifield procedure, recourseis made to a very porous polymeric resin, on which is immobilized thefirst C-terminal amino acid of the chain. This amino acid is immobilizedon a resin through its carboxyl group and its amine function isprotected. The amino acids, which are going to form the peptide chain,are thus immobilized, one after the other, on the amino group, which isdeprotected beforehand each time, of the portion of the peptide chainalready formed, and which is attached to the resin. When the whole ofthe desired peptide chain has been formed, the protective groups of thedifferent amino acids forming the peptide chain are eliminated and thepeptide is detached from the resin with the aid of an acid.

The invention additionally relates to hybrid polypeptides having atleast one polypeptide according to the invention, and a sequence of apolypeptide capable of inducing an immune response in man or animals.

Advantageously, the antigenic determinant is such that it is capable ofinducing a humoral and/or cellular response.

It will be possible for such a determinant to comprise a polypeptideaccording to the invention in glycosylated, pegylated, and/or otherwisepost-translationally modified form used with a view to obtainingimmunogenic compositions capable of inducing the synthesis of antibodiesdirected against multiple epitopes.

These hybrid molecules can be formed, in part, of a polypeptide carriermolecule or of fragments thereof according to the invention, associatedwith a possibly immunogenic part, in particular an epitope of thediphtheria toxin, the tetanus toxin, a surface antigen of the hepatitisB virus (patent FR 7921811), the VP1 antigen of the poliomyelitis virusor any other viral or bacterial toxin or antigen.

The procedures for synthesis of hybrid molecules encompass the methodsused in genetic engineering for constructing hybrid nucleotide sequencescoding for the polypeptide sequences sought. It will be possible, forexample, to refer advantageously to the technique for obtainment ofgenes coding for fusion proteins described by Minton in 1984.

The hybrid nucleotide sequences coding for a hybrid polypeptide as wellas the hybrid polypeptides according to the invention characterized inthat they are recombinant polypeptides obtained by the expression of thehybrid nucleotide sequences are likewise part of the invention.

The invention likewise comprises the vectors characterized in that theycontain one of the hybrid nucleotide sequences. The host cellstransformed by the vectors, the transgenic organisms comprising one ofthe transformed cells as well as the procedures for preparation ofrecombinant polypeptides using the vectors, the transformed cells and/orthe transgenic organisms are, of course, likewise part of the invention.

The polypeptides according to the invention, the antibodies according tothe invention described below and the nucleotide sequences according tothe invention can advantageously be employed in procedures for thedetection and/or identification of Alicyclobacillus acidocaldarius, in asample capable of containing them. These procedures, according to thespecificity of the polypeptides, the antibodies and the nucleotidesequences according to the invention, which will be used, will inparticular be able to detect and/or to identify Alicyclobacillusacidocaldarius.

The polypeptides according to the invention can advantageously beemployed in a procedure for the detection and/or the identification ofAlicyclobacillus acidocaldarius in a sample capable of containing them,characterized in that it comprises the following steps: a) contacting ofthis sample with a polypeptide or one of its fragments according to theinvention (under conditions allowing an immunological reaction betweenthe polypeptide and the antibodies possibly present in the biologicalsample); b) demonstration of the antigen-antibody complexes possiblyformed.

Any conventional procedure can be employed for carrying out such adetection of the antigen-antibody complexes possibly formed.

By way of example, a preferred method brings into play immunoenzymaticprocesses according to the ELISA technique, by immunofluorescence, orradioimmunological (RIA) processes or their equivalent.

Thus, the invention likewise relates to the polypeptides according tothe invention, labeled with the aid of an adequate label, such as, ofthe enzymatic, fluorescent or radioactive type.

Such methods comprise, for example, the following steps: deposition ofdetermined quantities of a polypeptide composition according to theinvention in the wells of a microtiter plate, introduction into thewells of increasing dilutions of serum, or of a biological sample otherthan that defined previously, having to be analyzed, incubation of themicrotiter plate, introduction into the wells of the microtiter plate oflabeled antibodies directed against pig immunoglobulins, the labeling ofthese antibodies having been carried out with the aid of an enzymeselected from those which are capable of hydrolyzing a substrate bymodifying the absorption of the radiation of the latter, at least at adetermined wavelength, for example at 550 nm, detection, by comparisonwith a control test, of the quantity of hydrolyzed substrate.

The polypeptides according to the invention allow monoclonal orpolyclonal antibodies to be prepared, which are characterized in thatthey specifically recognize the polypeptides according to the invention.It will advantageously be possible to prepare the monoclonal antibodiesfrom hybridomas according to the technique described by Köhler andMilstein in 1975. It will be possible to prepare the polyclonalantibodies, for example, by immunization of an animal, in particular amouse, with a polypeptide or a DNA, according to the invention,associated with an adjuvant of the immune response, and thenpurification of the specific antibodies contained in the serum of theimmunized animals on an affinity column on which the polypeptide, whichhas served as an antigen, has previously been immobilized. Thepolyclonal antibodies according to the invention can also be prepared bypurification, on an affinity column on which a polypeptide according tothe invention has previously been immobilized, of the antibodiescontained in the serum of an animal immunologically challenged byAlicyclobacillus acidocaldarius, or a polypeptide or fragment accordingto the invention.

The invention likewise relates to mono- or polyclonal antibodies ortheir fragments, or chimeric antibodies, characterized in that they arecapable of specifically recognizing a polypeptide according to theinvention.

It will likewise be possible for the antibodies of the invention to belabeled in the same manner as described previously for the nucleicprobes of the invention, such as a labeling of enzymatic, fluorescent orradioactive type.

The invention is additionally directed at a procedure for the detectionand/or identification of Alicyclobacillus acidocaldarius in a sample,characterized in that it comprises the following steps: a) contacting ofthe sample with a mono or polyclonal antibody according to the invention(under conditions allowing an immunological reaction between theantibodies and the polypeptides of Alicyclobacillus acidocaldariuspossibly present in the biological sample); b) demonstration of theantigen-antibody complex possibly formed.

The present invention likewise relates to a procedure for the detectionand/or the identification of Alicyclobacillus acidocaldarius in asample, characterized in that it employs a nucleotide sequence accordingto the invention.

More particularly, the invention relates to a procedure for thedetection and/or the identification of Alicyclobacillus acidocaldariusin a sample, characterized in that it contains the following steps: a)if need be, isolation of the DNA from the sample to be analyzed; b)specific amplification of the DNA of the sample with the aid of at leastone primer, or a pair of primers, according to the invention; c)demonstration of the amplification products.

These can be detected, for example, by the technique of molecularhybridization utilizing a nucleic probe according to the invention. Thisprobe will advantageously be labeled with a nonradioactive (cold probe)or radioactive isotope.

For the purposes of the present invention, “DNA of the biologicalsample” or “DNA contained in the biological sample” will be understoodas meaning either the DNA present in the biological sample considered,or possibly the cDNA obtained after the action of an enzyme of reversetranscriptase type on the RNA present in the biological sample.

A further embodiment of the invention comprises a method, characterizedin that it comprises the following steps: a) contacting of a nucleotideprobe according to the invention with a biological sample, the DNAcontained in the biological sample having, if need be, previously beenmade accessible to hybridization under conditions allowing thehybridization of the probe with the DNA of the sample; b) demonstrationof the hybrid formed between the nucleotide probe and the DNA of thebiological sample.

The present invention also relates to a procedure according to theinvention, characterized in that it comprises the following steps: a)contacting of a nucleotide probe immobilized on a support according tothe invention with a biological sample, the DNA of the sample having, ifneed be, previously been made accessible to hybridization, underconditions allowing the hybridization of the probe with the DNA of thesample; b) contacting of the hybrid formed between the nucleotide probeimmobilized on a support and the DNA contained in the biological sample,if need be after elimination of the DNA of the biological sample, whichhas not hybridized with the probe, with a nucleotide probe labeledaccording to the invention; c) demonstration of the novel hybrid formedin step b).

According to an advantageous embodiment of the procedure for detectionand/or identification defined previously, this is characterized in that,prior to step a), the DNA of the biological sample is first amplifiedwith the aid of at least one primer according to the invention.

Embodiments of methods include providing a recombinant, purified, and/orisolated polypeptide selected from the group consisting of a polypeptidehaving at least 90% sequence identity to SEQ ID NOS:1, 18, 35, 52, 69,86, 103, 120, 137, 154, 171, 188, 205, 222, 239, 256, 273, 290, 307,324, 341, 358, 375, 392, 409, 426, 443, 460, 477, 494, 511, 528, 545,562, 579, 596, 613, 630, 647, 664, 681, 698, 715, 732, 749, and 766 inor association with a cell membrane and transporting a sugar across thecell membrane using the a recombinant, purified, and/or isolatedpolypeptide in association with other cellular components.

Further embodiments of methods include placing a cell producing orencoding a recombinant, purified, and/or isolated nucleotide sequencecomprising a nucleotide sequence selected from the group consisting ofnucleotide sequences having at least 90% sequence identity to at leastone of the sequences of SEQ ID NOS:2, 19, 36, 53, 70, 87, 104, 121, 138,155, 172, 189, 206, 223, 240, 257, 274, 291, 308, 325, 342, 359, 376,393, 410, 427, 444, 461, 478, 495, 512, 529, 546, 563, 580, 597, 614,631, 648, 665, 682, 699, 716, 733, 750, and 767 and/or a recombinant,purified, and/or isolated polypeptide selected from the group consistingof a polypeptide having at least 90% sequence identity to at least oneof the sequences of SEQ ID NOS:1, 18, 35, 52, 69, 86, 103, 120, 137,154, 171, 188, 205, 222, 239, 256, 273, 290, 307, 324, 341, 358, 375,392, 409, 426, 443, 460, 477, 494, 511, 528, 545, 562, 579, 596, 613,630, 647, 664, 681, 698, 715, 732, 749, and 766 in a environmentcomprising temperatures at or above about 25, 30, 35, 40, 45, 50, 55,60, 65, 70, 75, 80, 85, 90, and/or 95 degrees Celsius and/or a pH at,below, and/or above 8, 7, 6, 5, 4, 3, 2, 1, and/or 0.

The present invention provides cells that have been geneticallymanipulated to have an altered capacity to produce expressed proteins.In particular, the present invention relates to Gram-positivemicroorganisms, such as Bacillus species having enhanced expression of aprotein of interest, wherein one or more chromosomal genes have beeninactivated, and/or wherein one or more chromosomal genes have beendeleted from the Bacillus chromosome. In some further embodiments, oneor more indigenous chromosomal regions have been deleted from acorresponding wild-type Bacillus host chromosome. In furtherembodiments, the Bacillus is an Alicyclobacillus sp. or Alicyclobacillusacidocaldarius.

In additional embodiments, methods of transporting sugars attemperatures at or above about 25, 30, 35, 40, 45, 50, 55, 60, 65, 70,75, 80, 85, 90, and/or 95 degrees Celsius and/or at a pH at, below,and/or above 8, 7, 6, 5, 4, 3, 2, 1, and/or 0 via a recombinant,purified, and/or isolated nucleotide sequence comprising a nucleotidesequence selected from the group consisting of nucleotide sequenceshaving at least 90% sequence identity to at least one of the sequencesof SEQ ID NOS:2, 19, 36, 53, 70, 87, 104, 121, 138, 155, 172, 189, 206,223, 240, 257, 274, 291, 308, 325, 342, 359, 376, 393, 410, 427, 444,461, 478, 495, 512, 529, 546, 563, 580, 597, 614, 631, 648, 665, 682,699, 716, 733, 750, and 767 and/or a recombinant, purified, and/orisolated polypeptide selected from the group consisting of a polypeptidehaving at least 90% sequence identity to at least one of the sequencesof SEQ ID NOS:1, 18, 35, 52, 69, 86, 103, 120, 137, 154, 171, 188, 205,222, 239, 256, 273, 290, 307, 324, 341, 358, 375, 392, 409, 426, 443,460, 477, 494, 511, 528, 545, 562, 579, 596, 613, 630, 647, 664, 681,698, 715, 732, 749, and 766.

In embodiments of the invention, any one of the isolated and/or purifiedpolypeptides, according to the invention, may be enzymatically orfunctionally active at temperatures at or above about 25, 30, 35, 40,45, 50, 55, 60, 65, 70, 75, 80, 85, 90, and/or 95 degrees Celsius and/ormay be enzymatically or functionally active at a pH at, below, and/orabove 8, 7, 6, 5, 4, 3, 2, 1, and/or 0. In further embodiments of theinvention, glycosylation, pegylation, and/or other post-translationalmodification may be required for the isolated and/or purifiedpolypeptides according to the invention to be enzymatically orfunctionally active at a pH at or below 8, 7, 6, 5, 4, 3, 2, 1, and/or 0or at temperatures at or above about 25, 30, 35, 40, 45, 50, 55, 60, 65,70, 75, 80, 85, 90, and/or 95 degrees Celsius.

The invention is described in additional detail in the followingillustrative examples. Although the examples may represent only selectedembodiments of the invention, it should be understood that the followingexamples are illustrative and not limiting.

EXAMPLES Example 1 Sugar Transport Using Nucleotide and Amino AcidSequences from Alicyclobacillus acidocaldarius

Provided in SEQ ID NOS:2, 19, 36, 53, 70, 87, 104, 121, 138, 155, 172,189, 206, 223, 240, 257, 274, 291, 308, 325, 342, 359, 376, 393, 410,427, 444, 461, 478, 495, 512, 529, 546, 563, 580, 597, 614, 631, 648,665, 682, 699, 716, 733, 750, and 767 are nucleotide sequences isolatedfrom Alicyclobacillus acidocaldarius and coding for the polypeptides ofSEQ ID NOS:1, 18, 35, 52, 69, 86, 103, 120, 137, 154, 171, 188, 205,222, 239, 256, 273, 290, 307, 324, 341, 358, 375, 392, 409, 426, 443,460, 477, 494, 511, 528, 545, 562, 579, 596, 613, 630, 647, 664, 681,698, 715, 732, 749, and 766, respectively. The nucleotide sequences ofSEQ ID NOS:2, 19, 36, 53, 70, 87, 104, 121, 138, 155, 172, 189, 206,223, 240, 257, 274, 291, 308, 325, 342, 359, 376, 393, 410, 427, 444,461, 478, 495, 512, 529, 546, 563, 580, 597, 614, 631, 648, 665, 682,699, 716, 733, 750, and 767 are placed into expression vectors usingtechniques standard in the art. The vectors are then provided to cellssuch as bacteria cells or eukaryotic cells such as Sf9 cells or CHOcells. In conjunction with the normal machinery present in the cells,the vectors comprising SEQ ID NOS:2, 19, 36, 53, 70, 87, 104, 121, 138,155, 172, 189, 206, 223, 240, 257, 274, 291, 308, 325, 342, 359, 376,393, 410, 427, 444, 461, 478, 495, 512, 529, 546, 563, 580, 597, 614,631, 648, 665, 682, 699, 716, 733, 750, and 767 produce the polypeptidesof SEQ ID NOS:1, 18, 35, 52, 69, 86, 103, 120, 137, 154, 171, 188, 205,222, 239, 256, 273, 290, 307, 324, 341, 358, 375, 392, 409, 426, 443,460, 477, 494, 511, 528, 545, 562, 579, 596, 613, 630, 647, 664, 681,698, 715, 732, 749, and 766. The polypeptides of SEQ ID NOS:1, 18, 35,52, 69, 86, 103, 120, 137, 154, 171, 188, 205, 222, 239, 256, 273, 290,307, 324, 341, 358, 375, 392, 409, 426, 443, 460, 477, 494, 511, 528,545, 562, 579, 596, 613, 630, 647, 664, 681, 698, 715, 732, 749, and 766are then isolated and/or purified. The isolated and/or purifiedpolypeptides of SEQ ID NOS:1, 18, 35, 52, 69, 86, 103, 120, 137, 154,171, 188, 205, 222, 239, 256, 273, 290, 307, 324, 341, 358, 375, 392,409, 426, 443, 460, 477, 494, 511, 528, 545, 562, 579, 596, 613, 630,647, 664, 681, 698, 715, 732, 749, and 766 are then each demonstrated tohave one or more of the activities provided in Table 1.

The isolated and/or purified polypeptides of SEQ ID NOS:1, 18, 35, 52,69, 86, 103, 120, 137, 154, 171, 188, 205, 222, 239, 256, 273, 290, 307,324, 341, 358, 375, 392, 409, 426, 443, 460, 477, 494, 511, 528, 545,562, 579, 596, 613, 630, 647, 664, 681, 698, 715, 732, 749, and 766 areplaced or transported into a cellular membrane and are demonstrated tohave activity in transporting sugars across the membrane in conjunctionwith other proteins or cellular components.

All references, including publications, patents, and patentapplications, cited herein are hereby incorporated by reference to thesame extent, as if each reference were individually and specificallyindicated to be incorporated by reference and were set forth in itsentirety herein.

While this invention has been described in certain embodiments, thepresent invention can be further modified within the spirit and scope ofthis disclosure. This application is therefore intended to cover anyvariations, uses, or adaptations of the invention using its generalprinciples. Further, this application is intended to cover suchdepartures from the present disclosure as come within known or customarypractice in the art to which this invention pertains and which fallwithin the limits of the appended claims and their legal equivalents.

BIBLIOGRAPHIC REFERENCES

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1. An isolated nucleic acid comprising a nucleic acid sequence encodinga polypeptide having at least 90% sequence identity to SEQ ID No: 137,wherein the polypeptide has Na⁺/melibiose symporter activity.
 2. Theisolated nucleic acid of claim 1, wherein the polypeptide has activityat about pH
 8. 3. The isolated nucleic acid of claim 1, wherein thepolypeptide has activity at a temperature at about 35 degrees Celsius.4. The isolated nucleic acid of claim 1, wherein the nucleic acid ispresent in a vector.
 5. The isolated nucleic acid of claim 1, whereinthe encoded polypeptide has activity below pH
 8. 6. The isolated nucleicacid of claim 1, wherein the encoded polypeptide has activity at atemperature above 35 degrees Celsius.
 7. The isolated nucleic acidaccording to claim 1, wherein said nucleic acid is as set forth in SEQID NO:138.
 8. A method of transporting a sugar across a cell membrane,the method comprising: providing to a cell the isolated nucleic acid ofclaim 1; incorporating into or associating with the cell membrane of thecell the protein encoded by the nucleic acid; and transporting a sugaracross said cell membrane utilizing the protein encoded by the nucleicacid.
 9. The method according to claim 8, wherein transporting a sugaracross said cell membrane utilizing the protein encoded by the nucleicacid occurs at about pH
 8. 10. The method according to claim 8, whereintransporting a sugar across said cell membrane utilizing the proteinencoded by the nucleic acid occurs at a temperature at about 35 degreesCelsius.
 11. The method according to claim 8, wherein moving a sugaracross said cell membrane utilizing the protein encoded by the nucleicacid occurs below pH
 8. 12. The method according to claim 8, whereinmoving a sugar across said cell membrane utilizing the protein encodedby the nucleic acid occurs at a temperature above 35 degrees Celsius.